Muscle myosin, a paradigm for molecular motors, is believed to generate force and movement by the rotation of a long alpha-helix in the head region (S1) that is stabilized by two light chain chains, ELC and RLC. How the hydrolysis of ATP in the catalytic domain is coupled to this motion remains poorly understood. Our overall aim is to elucidate the interactions occurring at the actomyosin interface, and determine how these interactions are transmitted to the LC-binding domain.. Techniques will include electron cryomicroscopy (cryoEM), and new methodologies in image analysis and computer-based fitting of atomic models into three dimensional reconstructions. Specific aim (1) will analyze the interactions of subdomains of the myosin head with actin the presence of MgADP and in its absence (rigor). S1 from fast and slow skeletal (beta- cardiac) myosins, and expressed smooth muscle constructs with mutations at the actin interface, will be compared to a completed cryoEM structure of smooth actoS1 to determine the generality of specific interactions and domain movements at the actomyosin interface, and their role in contraction. Specific aim (2) will extend this analysis to HMM, the two-headed subfragment of myosin, to determine the effect of the second head on the actomyosin complex will be determined by croEM and fluorescence spectroscopy using gold- and fluorescent probes, respectively, introduced into the N-terminal extension of the ELC, and near the phosphorylatable serine in the RLC. The last aim (3) is to determine the interaction between the two heads of myosin by preparing co-polymers of single- and double-headed myosins in a fluorescent rod filament for analysis by single-molecule mechanical studies.